For over a decade, the engineering of human muscle and cardiac tissue constructs has been an active area of innovation and development because of their enormous promise in the fields of regenerative medicine, drug development and basic research. A straightforward and direct approach to create tissue constructs would be to cut and maintain live tissue slices from explanted human hearts or tissue biopsies. This has been shown to be feasible in recent years from animal hearts, but for human tissue this approach faces enormous hurdles in terms of cellular damage created by cutting, short shelf life of cut tissues, limited availability and quantities of source material, and immunogenic responses.
The next best strategy is to create scaffolds onto which cells such as cardiac cells, for example, can be engrafted at the time of need. These scaffolds can be synthetic (derived from artificial materials such as polymers), or natural (derived from decellularized intact tissue). The latter are advantageous because they are biocompatible and possess mechanical and biochemical properties that are physiological, all of which have been difficult to reproduce so far in synthetic scaffolds Immunogenic responses are greatly muted, if not eliminated, with removal of the cellular component. Many methods have been developed to decellularize tissues from various organs, including the use of physical methods (e.g., freezing, pressure, tonicity), or chemical methods (acids, detergents, enzymes, chelators). In heart, decellularization has generally been applied to the whole organ to take advantage of the coronary vasculature as a means to effectively perfuse the tissue, beginning with a seminal study by Ott and colleagues (Ott, H. C. et al, Nature Medicine 14:213-21, 2008) that used SDS detergent, with alternative procedures by Wainwright and colleagues (Wainwright, J. M. et al, Tissue Engineering. Part C, Methods 16:525-532, 2010) using a complex hypotonic, hypertonic, enzyme, acid and detergent treatment. One study precut pig hearts into 3 mm thick slices prior to decellularization (Eitan, Y. et al., Tissue Engineering. Part C, Methods, 16(4), 671-83). In general, decellularization procedures leave behind an extracellular matrix scaffold that is biocompatible with low cell toxicity, and an ongoing challenge is to remove the residual cells without adversely affecting the matrix. The decellularized tissue can be dried and formed into an extracellular matrix powder, which can then be reconstituted as a gel in which cardiac cells can be suspended, grown and injected into the heart. Several companies have developed decellularized tissue products for clinical use, including: ACell (Columbia, Md.), marketing decellularized tissue matrix derived from urinary bladder of pigs, Ventrix (San Diego, Calif.), marketing an injectable gel created from extracellular matrix powder derived from pig hearts, Wright Medical Technology Inc. (Arlington, Tenn.), marketing acellular grafts from donated human skin for soft tissue replacement, Stryker (Kalamazoo, Mich.) marketing an acellular collagen membrane from human dermis for soft tissue repair, and Braun Melsungen AG (Melsungen, Germany), marketing a collagen implant from bovine pericardium for connective tissue substitution.
Cells grown in a 3D environment are widely acknowledged to experience important signaling cues that are lacking in conventional flat, 2D cultures. Four methods have already emerged to create relatively functional 3D cardiac tissue constructs: gels seeded with cardiac cells to create strands or rings of tissue, cardiac tissue patches created from stacks of cell sheets, scaffold-free clusters or patches created by aggregates of large numbers of cardiomyocytes, and compaction of cells into a mesh-like network. In general, only narrow strands or rings of gels seeded with cardiac cells have succeeded so far in producing an alignment of cells along a common axis, which is very important for efficient muscle function.
Therefore, there still exists an unmet need for 3D culture platforms and methods which even more closely recapitulate the in vivo microenvironment.